Apparatus for detecting atp in a liquid sample

ABSTRACT

An apparatus and kit for the detection of ATP in a liquid sample is provided. The apparatus and kit comprise a liquid reagent composition comprising luciferin and a sampling device having a sampling portion and a handling portion. The sampling portion is adapted to acquire and releasably retain a predetermined volume of a liquid sample in one or more cavity that is not substantially defined by space between a plurality of fibers. The sampling device comprises a dry coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having a pH of about 6.8 or lower, changes the pH of the liquid reagent composition to 6.9 or higher. A method of use of the apparatus or kit is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/668,520, filed Jul. 6, 2012, which is incorporatedherein by reference in its entirety.

BACKGROUND

Sampling programs are used to monitor critical raw materials, in-processmaterials, finished goods, and processing environments in the food andbeverage industry. Routine sampling and testing can allow qualityassurance personnel to detect undesirable materials, such asmicroorganisms, at a very early stage and take steps to preventsubsequent contamination of equipment and/or products. A variety oftests can be performed to detect the unwanted materials. Examples ofsuch tests include chemical residue tests (e.g., Adenosine triphosphate(ATP) bioluminescence tests and protein colorimetric tests), culturemethods, genetic tests (e.g., PCR), immunodiagnostic tests, andbioluminescent tests.

Sample-collection devices typically are used to collect surface samplesfor environmental tests. Commercially-available sample-collectiondevices include absorbent devices such as sponges, swabs, and the like.In addition, certain sample-collection devices are capable of collectinga predetermined volume of a liquid sample.

ATP is used routinely to detect a presence or absence of microorganismsin a sample. The chemical energy produced from the breakdown of ATP isconverted into light energy. Each molecule of ATP consumed in thereaction produces one photon of light. This light output can bequantified in a luminometer. The presence of ATP in a sample may be adirect indicator of the presence of a microorganism (i.e., the ATP isderived from a microorganism) or the ATP may be an indirect indicator ofthe presence of a microorganism (i.e., the ATP is derived fromvegetative or animal matter and indicates that nutrients that supportthe growth of microorganisms may be present in the sample). In addition,the presence or absence of ATP in a sample is used routinely to assessthe efficacy of cleaning processes in food, beverage, other industrialprocessed, healthcare (e.g. endoscopes) and for such as cooling andprocess waters and/or to determine whether biocide treatment has beeneffective in reducing the level of microorganisms.

SUMMARY

The present disclosure generally relates to the detection of ATP in asample using luciferase enzyme. In particular, the disclosure relates toa multi-purpose sampling device that comprises a dry coating comprisinga pH-adjusting reagent. The pH-adjusting reagent is capable of causing apH change when contacted with a liquid reagent composition comprisingluciferin. The pH change can facilitate the achievement of asubstantially steady-state light-emitting reaction in a shorter periodof time than at a lower pH. Surprisingly, the sampling device can becontacted with a liquid sample and, even though the dry coating may besoluble in the liquid sample, an effective amount of the pH-adjustingreagent is retained on and/or in the sampling device and the effectiveamount can be transferred to the liquid reagent composition where iteffects the aforementioned pH change. In addition, the sampling deviceobtains a predetermined volume of a liquid sample, thereby permittingthe operator to detect and, optionally, quantitate the amount of ATP inthe liquid sample.

In one aspect, the present disclosure provides a kit. The kit cancomprise a container with an opening and a cuvette portion that isadapted to be operationally coupled to a luminometer, a liquid reagentcomposition comprising luciferin, and a sampling device having asampling portion. The liquid reagent composition can be disposed in aclosed compartment. The pH of the liquid reagent composition is about6.8 or lower. The sampling portion of the sampling device is adapted toacquire and releasably retain a predetermined volume of a liquid samplein one or more cavity that is not substantially defined by space betweenfibers. The sampling device can comprise a dry coating that includes aneffective amount of a pH-adjusting reagent that, when contacted with theliquid reagent composition, changes the pH of the liquid reagentcomposition to 6.9 or higher.

In another aspect, the present disclosure provides an apparatus. Theapparatus can comprise a container with an opening and a cuvette portionthat is adapted to be operationally coupled to a luminometer, a liquidreagent composition comprising luciferin, and a sampling device having asampling portion. The liquid reagent composition can be disposed in aclosed compartment. The pH of the liquid reagent composition is about6.8 or lower. The sampling portion is adapted to acquire and releasablyretain a predetermined volume of a liquid sample in one or more cavitythat is not substantially defined by space between fibers. The samplingportion of the device comprises a dry coating that includes an effectiveamount of a pH-adjusting reagent that, when contacted with the liquidreagent composition, changes the pH of the liquid reagent composition to6.9 or higher.

In any embodiment of the above kit or apparatus, the pH-adjustingreagent can comprise a water-soluble reagent. In any of the aboveembodiments of the kit or the apparatus, the sampling portion cancomprise a calibrated loop. In any of the above embodiments of the kitor the apparatus, the closed compartment can comprise a frangible wall.In some embodiments, the frangible wall can be disposed in the containerbetween the opening and the cuvette portion. In some embodiments of thekit or the apparatus, the sampling device comprises the closedcompartment, wherein the sampling device further comprises a structurethat selectively releases the fluid composition from the container. Inany of the above embodiments of the kit or the apparatus, the samplingportion can comprise a foam material. In any of the above embodiments ofthe kit or the apparatus, the pH-adjusting reagent can comprise a buffercomponent selected from the group consisting of N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid],N-[tris(hydroxymethyl)methyl]glycine, and combinations thereof. In anyof the above embodiments of the kit or the apparatus, the pH-adjustingreagent can be coated in the one or more cavity. In any of the aboveembodiments of the kit or the apparatus, the coating further cancomprise an effective amount of a cell extractant. In any of the aboveembodiments of the kit or the apparatus, the luciferase enzyme canconsist essentially of a recombinant luciferase enzyme having luciferaseactivity that is less sensitive to variations in temperature, ionicdetergents, and reducing agents than a corresponding non-recombinantluciferase enzyme. In any of the above embodiments of the kit or theapparatus, the coating optionally does not comprise an effective amountof a phosphate buffer component. In any of the above embodiments of thekit or the apparatus, the predetermined volume can be about 0.01milliliters to about 0.25 milliliters.

In yet another aspect, the present disclosure provides a method. Themethod can comprise using a sampling device to obtain a predeterminedvolume of a liquid sample; contacting the predetermined volume of samplewith a liquid reagent composition in a container to form a reactionmixture; and using a luminometer to detect light emitted from thereaction mixture. The sampling device comprises a sampling portion. Thesampling portion is adapted to acquire and releasably retain apredetermined volume of liquid sample in one or more cavity. The one ormore cavity is not substantially defined by space between a plurality offibers. The sampling portion of the device comprises a dry coating thatcan include an effective amount of a pH-adjusting reagent that, whencontacted with a liquid reagent composition having a pH of about 6.8 orlower, changes the pH of the liquid reagent composition to 6.9 orhigher. The liquid reagent composition comprises a luciferin. Thecontainer comprises a cuvette portion adapted to be operationallycoupled to the luminometer.

In any embodiment of the above method, the method further can comprisethe step of agitating the predetermined volume of the sample and theliquid reagent composition in the container. In any of the aboveembodiments of the method, the liquid reagent composition further cancomprise a luciferase enzyme. In any of the above embodiments of themethod, wherein the luciferase enzyme activity can consist essentiallyof a recombinant luciferase enzyme having luciferase activity that isless sensitive to variations in temperature, ionic detergents, andreducing agents than a corresponding non-recombinant luciferase enzyme.In any of the above embodiments of the method, contacting the samplingdevice comprising the sample with liquid reagent composition cancomprise contacting the sampling device with the liquid reagentcomposition at a temperature within a range of 10° C. and 35° C.,inclusive. In any of the above embodiments of the method, aftercontacting the sampling device with liquid reagent composition, asubstantially steady-state amount of light is emitted from thecomposition in less than 20 seconds.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a cell extractant can beinterpreted to mean “one or more” cell extractants.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a side view of one embodiment of a sampling device accordingto the present disclosure.

FIG. 1a is a detailed side view of the sampling portion of the samplingdevice of FIG. 1.

FIG. 2 is a side view of an alternative embodiment of a sampling deviceaccording to the present disclosure.

FIG. 3 is a side view of another alternative embodiment of a samplingdevice according to the present disclosure.

FIG. 4 is an exploded view, partially in section, of one embodiment ofan apparatus according to the present disclosure.

FIG. 5 is a side view, partially in section, of the assembled apparatusof FIG. 4 showing the sampling device and the container in a firstoperational position with respect to each other.

FIG. 6 is a side view, partially in section, of the assembled apparatusof FIG. 4 showing the sampling device and the container in a secondoperational position with respect to each other.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. Unless specified or limitedotherwise, the terms “connected” and “coupled” and variations thereofare used broadly and encompass both direct and indirect connections andcouplings. Further, “connected” and “coupled” are not restricted tophysical or mechanical connections or couplings. It is to be understoodthat other embodiments may be utilized and structural or logical changesmay be made without departing from the scope of the present disclosure.Furthermore, terms such as “front,” “rear,” “top,” “bottom,” and thelike are only used to describe elements as they relate to one another,but are in no way meant to recite specific orientations of theapparatus, to indicate or imply necessary or required orientations ofthe apparatus, or to specify how the invention described herein will beused, mounted, displayed, or positioned in use.

The present disclosure generally relates to an apparatus and method fordetecting ATP from organic residues and/or cells in a sample. Accordingto the present disclosure, the ATP from cells is detected by contactingthe sample with a cell extractant to release ATP and detecting thereleased ATP using an enzymatic reaction that involves the reaction ofthe released ATP with luciferase enzyme activity in the presence ofluciferin and results in the production of measurable light.

In particular, the present disclosure provides an apparatus and/or a kitwith a sampling device that is adapted such that it 1) can acquire andreleasably retain a predetermined volume of liquid sample, 2) can usethe liquid sample to rehydrate an effective amount of dry, rehydratablepH-adjusting reagent, and 3) can deliver the liquid sample and effectiveamount of pH-adjusting reagent into a container in order to facilitate aluciferase-catalyzed light-emitting reaction. Advantageously, thepresent disclosure provides kits and apparatuses that can be used tostore aqueous mixtures comprising luciferin for extended periods of timeand instantly to adjust the pH of the mixtures in order to reduce thelag time required for a glow-type luciferase enzyme activity to achievestable light output (e.g., maximum light output) in the presence of ATPand luciferin, particularly at lower temperatures (e.g., temperaturesbelow 20° C.

FIG. 1 shows one embodiment of a sampling device that can be used in anapparatus, kit and/or method according to the present disclosure. Thesampling device 100 comprises a handling portion 20 and a samplingportion 30. In the illustrated embodiment, the sampling portion 30 iscoupled to the handling portion 20 via an optional stem 15. Typically,the sampling device 100 is shaped and dimensioned to be received in acorresponding container (not shown). The handling portion 20 can be madefrom a variety of materials (e.g., wood, metal, plastic, glass) usingprocesses known in the art and the handling portion 20 functions as alocation at which the sampling device 100 can be grasped and/ormanipulated during use. Optionally, the handling portion comprises ahandle 24 that is shaped and/or textured to facilitate manual ormechanical gripping of the sampling device 100. Optionally, the samplingdevice 100 further can comprise a piercing tip 40.

The sampling portion 30 may include any suitable structure that definesat least one cavity capable of obtaining (via capillary pressure) andretaining a liquid sample from a sample source. In one embodiment,sampling portion 30 defines a single cavity. In other embodiments, thesampling portion 30 includes a plurality of cavities that that are notin fluidic communication. Regardless of whether sampling portion 30comprises one or more cavity, the sampling portion 30 is designed toretain a predetermined maximum sample volume. The maximum sample volumemay be selected, for example, based on the desired sensitivity of thetest performed with the sample.

FIG. 1 shows one embodiment of a sampling portion 30 comprising aplurality of cavities 32. FIG. 1a shows a detailed view of the samplingportion 30 of FIG. 1. In the illustrated embodiment, the cavities 32consist of indentations that circle the sampling device 100. A personhaving ordinary skill in the art will recognize the predetermined volumecan be achieved using a variety of design configurations for the one ormore cavity. For example, International Publication No. WO 2009/134509,which is incorporated herein by reference in its entirety, discloses avariety of sample acquisition devices comprising cavities that aresuitable for obtaining and retaining a predetermined volume of liquidsample.

In an alternative embodiment, the sampling portion of a sampling deviceof the present disclosure may comprise a plurality of cavities formed ina foam material. Foam articles (e.g., sponges, sponges mounted on ahandle) for obtaining samples from environmental surfaces are known inthe art. FIG. 2 shows a sampling device 100′ comprising a samplingportion 30 that comprises a foam material. The foam material comprisesindividual cells or void spaces that are capable of obtaining andretaining a liquid sample. Suitable foam materials for use in a samplingdevice of the present disclosure include, for example, polyurethanefoams, polyethylene foams, and polystyrene foams. In some embodiments,the foams may be treated (e.g., corona-treated or electron beam-treated)in order to make the surface of the polymer more hydrophilic. The foammaterial can be coupled to a stem 15 and/or a handling portion 20 usingmaterials (e.g., adhesives, mechanical fasteners, or the like) andprocesses known in the art.

FIG. 3 shows yet another alternative embodiment of a sampling device100″ according to the present disclosure. In this embodiment, thesampling portion 30 comprises a calibrated loop 34 configured to obtainand retain a predetermined volume of sample liquid. Calibrated loops areknown in the art and the loop may take a variety of forms including, forexample, a circular form (as shown in FIG. 3) or an obround form.Optionally, any sampling device according to the present disclosurefurther may comprise a coating area on which a coating comprising apH-adjusting reagent according to the present disclosure can bedisposed. An embodiment of a sampling device 100″ comprising a coatingarea 36 onto which a coating can be applied is shown in FIG. 3. Thecoating can be comprise an effective amount of a pH-adjusting reagentthat, when contacted (e.g., mixed) with a liquid reagent compositionaccording to the present disclosure, adjusts the pH of the liquidreagent composition having an initial pH of 6.8 or lower to an adjustedpH of 6.9 or higher, preferably to an adjusted pH of about pH 7.2-8.0,more preferably to an adjusted pH of about pH 7.2-7.8. Also shown inFIG. 3 are the handling portion 20 comprising a handle 24, the stem 15,and piercing tip 40. In any embodiment, the pH-adjusting reagent can bea water-soluble reagent.

In some embodiments, sampling portion 30 is formed of a material havinga surface energy in a range of about 20 dynes/centimeter (dyn/cm) toabout 82 dyn/cm, such as about 45 dyn/cm to about 72 dyn/cm. In someembodiments, the material for sampling portion 30 is selected to have asurface energy close to that of water, or about 72 dyn/cm. A sample maybe easier to remove from sampling portion 30 compared to a conventionalmedical swab that includes a fibrous tip because the sample is heldwithin sampling portion 30 by surface energy, rather than absorption, asis the case with some conventional medical swabs. That is, less energymay be required to remove sample liquid and/or liquid-suspendedparticles from sampling portion 30. In some cases, a large percentage ofsample particles are removed from sampling portion 30 without the aid ofa machine vortexer, although a machine vortexer may be utilized to helpelute the sample from sampling portion 30.

In general, sampling portion 30 is comprised of a material that achievesthe desired sample acquisition characteristics, which may depend uponthe type of sample acquired (e.g., some liquid samples may includedissolved or suspended solids that affect the surface tension of theliquid). Material properties that may affect the ability of samplingportion 30 to acquire and retain a sample include, for example, surfaceenergy. For example, as described above, the material may be selected tohave a particular surface energy in order to draw the sample into theone or more cavity defined by sampling portion 30 by capillary force.Other characteristics of the material from which the sampling portion ismade may include substantial inertness relative to the sample or arelatively low rate of elution of chemicals or other contaminants thatmay affect luciferase enzyme activity, e.g., when the sample is releasedfrom sampling portion 30.

In some embodiments, sampling portion 30 may include a base materialthat does not necessarily include the desired sample acquisitioncharacteristics, and an external layer (e.g., a coating) comprising amaterial that affords hydrophilic, hydrophobic, positively-charged ornegatively-charged surfaces to achieve the desired sample acquisitioncharacteristics. For example, an inorganic coating (e.g., a silicacoating) or an organic coating (e.g., polymeric coatings, such aspolyacrylate) may afford hydrophilic characteristics to sampling portion30. Surface energy (or surface tension) characteristics of a materialforming sampling portion 30 may also be achieved with the aid ofphysical treatments, such as, but not limited to, corona treating inwhich the material being treated is exposed to an electrical discharge,or corona, electron beam treatments.

In some embodiments, the sampling portion 30 may be formed at least inpart of relatively rigid polymer (e.g., nylon, polysulfone,polycarbonate, or combinations thereof) or it may be formed using a morecompliant polymer, such as silicone. Example materials for samplingportion 30 include, but are not limited to, any thermoplastic materialssuitable for casting, profile extrusion, molding (e.g., injectionmolding) or embossing including, for example, polyolefins, polyesters,polyamides, poly(vinyl chloride), polymethyl methacrylate,polycarbonate, nylon, and the like. In other embodiments, samplingportion 30 may be formed by molding or embossing a sheet of suitablematerial into the desired cavity structure.

A sampling device of the present disclosure comprises a dry coating. Inany embodiment, the dry coating can be disposed on and/or in (e.g., in acavity) the sampling portion of the device so that, when the device isdipped into the liquid sample, the coating is contacted with the liquidsample. In any embodiment, at least a part of the dry coating can bedisposed on another portion (e.g., the stem, the piercing tip) of thesampling device. The dry coating includes an effective amount of apH-adjusting reagent that, when contacted (e.g. mixed) with a liquidreagent composition of the present disclosure, changes the pH of theliquid reagent composition. In any embodiment, the pH-adjusting reagentcomprises a water-soluble reagent. In any embodiment, the pH-adjustingreagent comprises a component of a buffer solution (e.g. thepH-adjusting reagent comprises a base or a conjugate acid) having a pKaof 7.0 or higher, preferably having a pKa of 7.2 or higher. pH-adjustingreagents that each comprise a suitable buffer component include, but arenot limited to, N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonicacid], N-[tris(hydroxymethyl)methyl]glycine, and combinations thereof.In some embodiments, the coating does not comprise an effective amountof a pH-adjusting reagent comprising phosphate. A pH-adjusting reagentcomprising an amount of phosphate effective to change the pH of theliquid reagent composition from an initial pH 6.8 or lower to anadjusted pH of 6.9 or higher may also be sufficient to at leastpartially inhibit luciferase enzyme activity. In any embodiment, thepH-adjusting reagent comprises a metallic base (e.g., sodium hydroxide,potassium hydroxide).

In any embodiment, the coating further can comprise a cell lysis agentthat does not substantially inhibit luciferase enzyme activity. Examplesof suitable cell lysis agents include, but are not limited to,chlorhexidine digluconate, TRITON X-100, lysozyme, lysostaphin,bacteriophage lysin, a quaternary ammonium compound (e.g., benzalkoniumchloride, benzethonium chloride), cetyl trimethylammonium chloride(CTAB), dodecyltrimethylammonium chloride (DTAB), TWEEN 20, TWEEN 80,and a combination of any two or more of the foregoing.

The amount of pH-adjusting reagent coated onto the sampling deviceshould be enough to change the pH of the liquid reagent composition froman initial (i.e., before contact with the pH-adjusting reagent) pH of6.8 or lower to an adjusted pH of about 6.9 or higher (after contactwith the pH-adjusting reagent). Preferably, the amount of pH-adjustingreagent coated on the sampling device should be enough to change the pHof the liquid reagent composition from an initial pH of 6.8 or lower toa pH about 7.2 or higher.

The coating can be applied to the sampling device using any suitablecoating method. In any embodiment, the coating initially can be appliedto the sampling device as a liquid coating (i.e., the pH-adjustingreagent is dissolved or suspended in a solvent such as water, forexample). The solvent is subsequently dried to leave the substantiallydry pH-adjusting reagent coated on the sampling device. In anyembodiment, the liquid coating is applied by dipping the sampling device(e.g., the sampling portion of the sampling device) into a liquidcomprising the pH-adjusting reagent. While immersed in the coatingliquid, the sampling device can be agitated to facilitate movement ofthe coating liquid into the one or more cavity of the sampling portion.After coating the device, the coated portion can be dried (e.g., in astream of air, a convection oven, or the like) under conditions (e.g.,ambient or above-ambient (warm) temperatures) sufficient to evaporatethe solvent without substantially degrading the pH-adjusting reagentand/or cell lysis agent.

Kits and apparatuses of the present disclosure further comprise acontainer. The container functions as a vessel in which to contact asample with the liquid reagent composition described herein. Thecontainer comprises an opening and is configured (e.g., shaped anddimensioned) to receive at least a portion (e.g., the sampling portion)of the sampling device. FIG. 4 shows an exploded view, partially insection, of one embodiment of an apparatus comprising a containeraccording to the present disclosure. The apparatus 1000 comprises asampling device 100 comprising a handling portion 20 with a handle 24and a sampling portion 30 according to any of the embodiments describedherein. The apparatus 1000 further comprises a container 200, shown incross-section in FIG. 4.

The container comprises at least one wall 50 and an opening 52, theopening configured to receive the sampling device 100. As discussedbelow, the apparatus 1000 with the opening 52 shown in the illustratedembodiment of FIG. 4 is shaped and dimensioned to receive the entiresampling device 100. The container further comprises a cuvette portion54 that is adapted to be operationally coupled to a luminometer.Typically, the cuvette portion 54 is operationally coupled to aluminometer by placing the cuvette portion 54 of the container 200, orthe entire container 200, into a complementary-shaped receivingcompartment of the luminometer. The cuvette portion 54 is fabricatedfrom optically-transmissible material (e.g., glass or a polymericmaterial such as polyethylene, polypropylene, polycarbonate, orpolystyrene, for example) that permits the transmission there through oflight emitted as a product of a reaction catalyzed by luciferase enzyme.

The container 200 may be fabricated as a unitary article (not shown),for example, using extrusion and/or molding processes known in the art.Alternatively, portions of the container 200, such as thecylindrically-shaped sleeve 56 and the cuvette 55 of the illustratedembodiment of FIG. 4, may be fabricated separately and coupled togetherusing suitable means (e.g., an adhesive, a heat sealing process, a sonicweld, and/or by press-fitting the sleeve 56 and cuvette 55 together).The sleeve 56 may be fabricated via a molding or extrusion process, forexample, using a plastic polymer such as polypropylene or polyethylene,for example. The cuvette 55 can be formed in a variety of geometricshapes, such as cubic, cuboid, cylindrical, conical, frusto-conical,other suitable geometric shapes, and combinations thereof. Preferably,the walls of the cuvette 55 are configured to allow the passage of light(e.g., visible light) into and/or out of the cuvette 55. Optionally, thecontainer further may comprise a lamina 60. The lamina 60 can hold thecuvette 55 firmly together with the sleeve 56. The lamina 60 can be madefrom paper or a plastic film, for example, and may be used as a label.

Kits and apparatuses of the present disclosure further comprise a liquidreagent composition (liquid reagent composition 70 shown in FIGS. 4-6).The liquid reagent composition comprises luciferin at a concentration(e.g., 0.3-0.4 mg/L luciferin) sufficient to facilitate a light-emittingreaction in the presence of luciferase; preferably, a glow-typeluciferase; and a source of ATP. In a method of the present disclosure,the source of ATP can be a sample as disclosed herein or a solutioncontaining a predefined amount or concentration of ATP (i.e., a positivecontrol or an ATP standard). In any embodiment, the liquid reagentcomposition can comprise an aqueous solution. The liquid reagentcomposition used in the apparatus or kit of the present disclosure hasan initial (i.e., before contact with the pH-adjusting reagent) pH of6.8 or lower. Preferably, liquid reagent composition has an initial(i.e., before contact with the pH-adjusting reagent) pH of 6.4 or lower.

In any embodiment, the liquid reagent composition optionally maycomprise a buffering agent. Suitable buffering agents have a pKa that iseffective to maintain the pH of the liquid reagent composition at a pHof 6.8 or lower, preferably at a pH of 6.4 or lower.N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimiting example of asuitable buffering agent. The buffering agent should be present in theliquid reagent composition in an amount (e.g., about 16 mM ADA in 425microliters of liquid reagent composition at pH 6.4-6.8) that is highenough to effectively maintain the pH of the liquid reagent composition,yet low enough so that it does not substantially resist a change in thepH of the liquid reagent composition mediated by the pH-adjustingreagent coated on the sampling portion of the sampling device. In anyembodiment, the liquid reagent composition further may comprise aluciferase enzyme. In any embodiment, the luciferase enzyme can consistessentially of a recombinant luciferase enzyme having luciferaseactivity that is less sensitive to variations in temperature, ionicdetergents, and reducing agents than a corresponding non-recombinantluciferase enzyme. The luciferase enzyme can be present in the liquidreagent composition at a concentration of about 9 mg/L, for example. Inany embodiment, the liquid reagent composition further can comprise asource of magnesium ions (e.g., magnesium diacetate tetrahydrate). Thesource of magnesium ions can be present at a concentration (e.g., about3 mM, for example) that does not substantially interfere with luciferaseenzyme activity when the liquid reagent composition is mixed with theliquid sample. In any embodiment, the liquid reagent composition furthercan comprise a protein (e.g., bovine serum albumin). Without being boundby theory, the protein may stabilize the luciferase enzyme duringprolonged storage and/or make the luciferase enzyme less susceptible toprotease enzyme activities. At its working concentration, the proteinshould not substantially interfere with luciferase enzyme activity whenthe liquid reagent composition is mixed with the liquid sample. Theprotein may be present in the liquid reagent composition at aconcentration of about 0.046 weight percent, for example. In anyembodiment, the liquid reagent composition further can comprise apreservative (e.g., 0.046 weight percent sodium azide) at aconcentration that does not substantially interfere with luciferaseenzyme activity when the liquid reagent composition is mixed with theliquid sample. In any embodiment, the liquid reagent composition furthermay comprise ethylenediamine tetraacetic acid (EDTA). Without beingbound by theory, the EDTA can act as a preservative in the liquidreagent composition and may also function to chelate ions present in theliquid reagent composition and/or the liquid sample.

In any embodiment, the liquid reagent composition can be disposed in aclosed compartment. In some embodiments, the closed compartmentcomprises a frangible wall. In the illustrated embodiment of FIG. 4, thecontainer further comprises a liquid-resistant frangible wall 65disposed proximate the cuvette 55 that forms a compartment 67. Thefrangible wall 65 can be made from a water-resistant material, such asplastic film, metal foil, or a metal-coated plastic film, for example.The frangible wall 65 can be coupled to the cuvette 55 and/or the sleeve56 via an adhesive, a heat seal, a sonic weld, or other means known inthe art to form a liquid-resistant seal to retain the liquid reagentcomposition 70 in the compartment 67.

In an alternative embodiment (not shown), the liquid reagent compositionmay be disposed in a frangible ampoule (e.g., a glass or polymericampoule) which is disposed in the container. The liquid reagentcomposition can be released from the frangible ampoule by squeezing thecontainer with enough force to fracture the ampoule or by urging thesampling device (e.g., the piercing tip of the sampling device) againstthe ampoule with enough force to fracture the ampoule.

In a further embodiment (not shown), the liquid reagent composition maybe disposed in a compartment disposed in the sampling device. Nason(U.S. Pat. No. 5,266,266, which is incorporated herein by reference inits entirety) discloses a swab device comprising a hollow stem and aliquid-containing closed compartment with an actuatable valve (break-offnib) that releases the liquid into the hollow stem for delivery intotube in which at least a portion of the swab is disposed. Any samplingdevice of the present disclosure may be modified to include the hollowstem and reagent chamber of Nason. The reagent chamber can contain theliquid reagent composition of the present disclosure and the liquidreagent composition can be transferred into the container of the presentdisclosure using the technique described by Nason. The liquid reagentcomposition can be transferred to the container before or after theliquid sample is acquired using the sampling device according to thepresent disclosure. Other compartments for temporarily storing liquidreagent compositions to be used in a reaction (e.g., a chemical and/orenzymatic reaction) are known in the art and a person of ordinary skillin the art will recognize such compartments can be used to temporarilycontain the liquid reagent composition of the present disclosure.Non-limiting examples of such compartments can be found in U.S. Pat.Nos. 7,399,984; 6,548,018; 5,965,453; and 6,524,530, which are allincorporated herein by reference in their entirety.

The present disclosure provides a kit. The kit may be used, for example,to detect a presence or absence of ATP in a liquid sample according themethod described herein. In any embodiment, the kit may compriseinstructions disclosing an embodiment of the method disclosed herein.

Kits of the present disclosure comprise a container according to any ofthe embodiments disclosed herein. The container comprises an opening anda cuvette portion that is adapted to be operationally coupled to aluminometer. Kits of the present disclosure further comprise a liquidreagent composition comprising luciferin according to any of theembodiments disclosed herein. The initial pH (i.e., before contact witha pH-adjusting reagent of the present disclosure) of the liquid reagentcomposition is about pH 6.8 or lower; preferably, about pH 6.4 or lower.Kits of the present disclosure further comprise a sampling device havinga sampling portion and a handling portion according to any of theembodiments disclosed herein. The liquid reagent composition is disposedin a closed compartment disposed in the container or the samplingdevice, as disclosed herein. The sampling portion of the sampling deviceis adapted to acquire and releasably retain a predetermined volume of aliquid sample, as described herein, in one or more cavity that is notsubstantially defined by space between a plurality of fibers. Thesampling portion of the sampling device comprises a dry coating thatincludes an effective amount of a pH-adjusting reagent that, whencontacted (e.g., mixed) with the liquid reagent composition, changes thepH of the liquid reagent composition to 6.9 or higher, preferably toabout pH 7.2-8.0, more preferably to about pH 7.2-7.8. In anyembodiment, the pH-adjusting reagent comprises a water-soluble reagent.

Kits and apparatuses of the present disclosure can be used in a methodof detecting a presence or an absence of ATP in a liquid sample. Thepresence of ATP in a sample can indicate a possible presence of organicresidues and/or microorganisms (e.g., pathogenic microorganisms) in thesample. Moreover, the quantity of ATP in the sample can be an indicatorof the relative number of microorganisms in the sample. Therefore, it isdesirable to test a predetermined volume of sample, so that the amountof ATP per unit volume of sample can be determined. The amount of ATPper unit volume of sample can be compared to a predetermined value todetermine whether the sample is acceptable for use in a particularapplication (e.g., food or beverage production), whether a cleaningprocess has been effective in removing organic residues, and/or whethera biocide treatment has been effective in reducing the number ofmicroorganisms.

In one embodiment, the method comprises using a sampling device toobtain a predetermined volume of liquid sample, contacting thepredetermined volume of sample with a liquid reagent composition in acontainer to form a reaction mixture, and using a luminometer to detectlight emitted from the reaction mixture. The predetermined volume ofliquid sample can be about 10 microliters to about 250 microliters. Insome embodiments, the predetermined volume of liquid sample can be about100 microliters to about 200 microliters. In some embodiments, thepredetermined volume of liquid sample can be about 125 microliters toabout 175 microliters.

The sample comprises a liquid. The liquid may comprise water. The samplefurther may comprise solids that are dissolved, dispersed, and/orsuspended in the liquid. Nonlimiting examples of suitable samplesinclude clean-in-place (CIP) rinse-water samples, process and coolingwater samples, and endoscope rinse water samples.

The sampling device used in the method of the present disclosure can beany sampling device disclosed herein. The sampling device comprises asampling portion and a handling portion. The sampling portion is adaptedto acquire and releasably retain a predetermined volume of liquid samplein one or more cavity. The one or more cavity is not substantiallydefined by space between a plurality of fibers. The sampling portion ofthe device comprises a dry coating that includes an effective amount ofa pH-adjusting reagent that, when contacted (e.g., mixed) with a liquidreagent composition having an initial pH of 6.8 or lower, adjusts the pHof the liquid reagent composition to 6.9 or higher.

The liquid reagent composition used in the method of the presentdisclosure comprises a luciferin (e.g., firefly luciferin). Luciferin isa compound that undergoes oxidation by luciferase enzyme activity toproduce oxyluciferin and light. Luciferin compounds can be unstable whenstored for extended periods of time in liquid solutions at a pH above6.8. Thus, the liquid reagent composition used in the method of thepresent disclosure has an initial pH of 6.8 or lower. Preferably, liquidreagent composition has an initial pH of 6.4 or lower.

In any embodiment, the liquid reagent composition may further comprise aluciferase enzyme. In a preferred embodiment, the luciferase enzymecomprises a recombinant luciferase enzyme having luciferase activitythat is less sensitive than a corresponding non-recombinant luciferaseenzyme to variations in temperature, ionic detergents, and reducingagents. In some embodiments, the luciferase enzyme consists essentiallyof a recombinant luciferase enzyme having luciferase activity that isless sensitive to variations in temperature, ionic detergents, andreducing agents than a corresponding non-recombinant luciferase enzyme.

In any embodiment, the liquid reagent composition optionally maycomprise a buffering agent. The buffering agent can be used to maintainthe liquid reagent composition at a pH (e.g., ≤6.8) that is suitable tostore a liquid solution containing luciferin.N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimiting example of asuitable buffering agent. The buffering agent should be present in theliquid reagent composition in an amount (e.g., about 16 mM ADA in 425microliters of liquid reagent composition at pH 6.4-6.8) that is highenough to effectively maintain the pH of the liquid reagent composition,yet low enough so that it does not substantially resist a change in thepH of the liquid reagent composition mediated by the pH-adjustingreagent coated on the sampling portion of the sampling device.

The container used in the method of the present disclosure comprises acuvette portion. The cuvette portion permits the transmission therethrough of light emitted as a product of the reaction catalyzed byluciferase enzyme. The cuvette portion is adapted (e.g., shaped anddimensioned) to be operationally coupled to the luminometer. In someembodiments, using a luminometer to detect light emitted from thereaction mixture comprises detecting a presence or an absence of ATP inthe liquid sample. In some embodiments, using a luminometer to detectlight emitted from the reaction mixture comprises measuring a quantity(e.g., a relative quantity or an absolute quantity) of ATP present inthe liquid sample.

Contacting the predetermined volume of sample with a liquid reagentcomposition in a container to form a reaction mixture comprises bringingthe sampling portion of the sampling device into contact with the liquidreagent composition in a container. The contact may be achieved, forexample, by immersing at least a part; preferably, the entire samplingportion; of the sampling device in the liquid reagent composition. Thecontact forms a reaction mixture (e.g., by diffusion of at least part ofthe liquid sample into the liquid reagent composition). If it is notalready present in the liquid reagent composition, luciferase can beadded to the reaction mixture. Thus, if ATP is present in the liquidsample, the ATP can facilitate a light-emitting reaction catalyzed byluciferase enzyme. The light-emitting reaction can be detected in theaforementioned luminometer.

FIGS. 5 and 6 illustrate one embodiment of a process of contacting apredetermined volume of sample with a liquid reagent composition in acontainer to form a reaction mixture. After obtaining the predeterminedvolume of liquid sample using the sampling device 100, the samplingdevice is inserted into the container 200. FIG. 5 shows the samplingdevice 100 with the stem 15 and sampling portion 30 in the container. Inthis configuration, the sampling device 100 and container 200 aredisposed in a first operational position with respect to one another andthe apparatus 1000 is ready to bring the liquid sample into contact withthe liquid reagent composition 70 located in the compartment 67. Usingmanual or mechanical pressure against the handle 24 of the samplingdevice 100, the piercing tip 40 is urged toward the compartment 67 untilit pierces the frangible wall 65 and brings the liquid sample (notshown) associated with the sampling portion 30 into contact with theliquid reagent composition 70, as shown in FIG. 6. In the illustratedembodiment of FIG. 6, the sampling device 100 and container 200 aredisposed in a second operational position with respect to each other. Inthis embodiment, the sampling device 100 is fully-inserted into thecontainer 200 and the sampling portion 30 of the sampling device iscontacting the liquid reagent composition 70.

In any embodiment, contacting the predetermined volume of sample with aliquid reagent composition in a container to form a reaction mixturefurther can comprise the step of agitating the sample and the liquidreagent composition in the container. This may be done manually ormechanically, for example, by rapidly shaking the container in aswirling motion, a pendulum-like motion, or by contacting the containerwith a machine that vibrates the container.

The cuvette portion of the container is operationally coupled to theluminometer in order to detect the light emission resulting fromluciferase enzyme activity. Commercially-available luminometers aresuitable for use in any embodiment of the method of the presentdisclosure. A non-limiting example of a suitable luminometer is the 3M™Clean-Trace™ NG Luminometer commercially available from 3M Company, St.Paul, Minn. Typically, the cuvette is operationally coupled by placing aportion (e.g., the cuvette portion) of the container or the entirecontainer into a corresponding receiving compartment of the luminometer.The cuvette portion may be operationally coupled to the luminometerbefore or after forming the reaction mixture. Typically, the cuvetteportion is operationally coupled to the luminometer after the reactionmix is formed. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 5 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 10 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 15 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 20 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 30 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 45 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 60 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 90 seconds after forming the reactionmixture. In some embodiments the cuvette portion is operationallycoupled to the luminometer up to 2 minutes after forming the reactionmixture. Preferably, the cuvette is operationally coupled to theluminometer within about 5 to 10 seconds after forming the reactionmixture.

After operationally coupling the cuvette portion to the luminometer, thelight-emission is monitored by the luminometer. After the reactionmixture is formed, it may take a period of time for the reaction toreach a relatively stable quantity of light emission. This phenomenon isillustrated by the data in Tables 4-9. The length of time for the lightemission to reach a relatively stable plateau is inversely related tothe temperature of the reaction, as summarized in Table 11.

In any embodiment of the method, the liquid reagent compositioncomprises luciferin at a concentration (e.g., about 0.3-0.4 mg/Lluciferin) sufficient to facilitate a light-emitting reaction in thepresence of luciferase and a source of ATP. In the method of the presentdisclosure, the source of ATP can be a liquid sample as disclosed hereinor a solution containing a predefined amount or concentration of ATP(i.e., a positive control or an ATP standard). In any embodiment, theliquid reagent composition can comprise an aqueous solution. The liquidreagent composition used in the apparatus or kit of the presentdisclosure has an initial pH (i.e., before contact with a pH-adjustingreagent of the present disclosure) of 6.8 or lower. Preferably, liquidreagent composition has an initial pH of 6.4 or lower.

In any embodiment of the method, the liquid reagent compositionoptionally may comprise a buffering agent. Suitable buffering agentshave a pKa that is effective to maintain the initial pH of the liquidreagent composition at a pH of 6.8 or lower, preferably at a pH of 6.4or lower. N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimitingexample of a suitable buffering agent. The buffering agent should bepresent in the liquid reagent composition in an amount (e.g., about 16mM ADA in 425 microliters of liquid reagent composition at pH 6.4-6.8)that is high enough to effectively maintain the pH of the liquid reagentcomposition, yet low enough so that it does not substantially resist achange in the pH of the liquid reagent composition mediated by thepH-adjusting reagent coated on the sampling portion of the samplingdevice. In any embodiment of the method, the liquid reagent compositionfurther may comprise a luciferase enzyme. In any embodiment of themethod, the luciferase enzyme can consist essentially of a recombinantluciferase enzyme having luciferase activity that is less sensitive tovariations in temperature, ionic detergents, and reducing agents than acorresponding non-recombinant luciferase enzyme. The luciferase enzymecan be present in the liquid reagent composition at a concentration ofabout 9 mg/L, for example. In any embodiment of the method, the liquidreagent composition further can comprise a source of magnesium ions(e.g., magnesium diacetate tetrahydrate). The source of magnesium ionscan be present at a concentration (e.g., about 3 mM, for example) thatdoes not substantially interfere with luciferase enzyme activity whenthe liquid reagent composition is mixed with the liquid sample. In anyembodiment of the method, the liquid reagent composition further cancomprise a protein (e.g., bovine serum albumin). The protein may bepresent in the liquid reagent composition at a concentration of about0.046 weight percent, for example. In any embodiment of the method, theliquid reagent composition further can comprise a preservative (e.g.,0.046 weight percent sodium azide) at a concentration that does notsubstantially interfere with luciferase enzyme activity when the liquidreagent composition is mixed with the liquid sample.

In any embodiment of the method of the present disclosure, contactingthe sampling device comprising the liquid sample with liquid reagentcomposition comprises contacting the sampling device with the liquidreagent composition at a particular temperature (e.g. ambienttemperature). In general, when the contact occurs at lower temperatures,a longer period of time is required for the light-emitting reaction toreach a relatively stable amount of light emission. Conversely, when thecontact occurs at higher temperatures, a shorter period of time isrequired for the light-emitting reaction to reach a relatively stableamount of light emission. In any embodiment of the method of the presentdisclosure, contacting the sampling device comprising the liquid samplewith liquid reagent composition comprises contacting the liquid samplewith the liquid reagent composition at temperature within a range of 10°C. and 35° C., inclusive. Preferably, contacting the sampling devicecomprising the liquid sample with liquid reagent composition comprisescontacting the liquid sample with the liquid reagent composition attemperature within a range of 15° C. and 30° C., inclusive.

In any embodiment of the method, after contacting the sampling devicewith the liquid reagent composition, a substantially steady-state amountof light is emitted from the composition after a period of time. Asdiscussed above, the period of time may be affected by the temperatureat which the contact occurs. In any embodiment of the presentdisclosure, a substantially steady-state amount of light is emitted fromthe composition in less than 25 seconds (e.g., when contact occurs at atemperature of about 10° C. to about 20° C.). In any embodiment of thepresent disclosure, a substantially steady-state amount of light isemitted from the composition in about 22 seconds or less (e.g., whencontact occurs at a temperature of about 10° C. to about 20° C.). Insome embodiments, a substantially steady-state amount of light isemitted from the composition in less than 20 seconds (e.g., when contactoccurs at a temperature of about 14° C. to about 20° C.). In someembodiments, a substantially steady-state amount of light is emittedfrom the composition in about 10 seconds or less (e.g., when contactoccurs at a temperature of about 20° C.).

The method of the present invention substantially reduces the amount oftime necessary for a luciferase enzyme, in contact with a samplecomprising ATP and a liquid reagent composition according to the presentdisclosure, to catalyze a light-emitting reaction that is substantiallysteady state. Table 11 (below) shows the mean amount of time requiredfor the reaction to reach a substantially steady-state light emission isreduced about 3-fold by the method of the present disclosure attemperatures between 10° C. and 20° C., inclusive.

EMBODIMENTS

Embodiment A is a kit, comprising:

a container with an opening and a cuvette portion that is adapted to beoperationally coupled to a luminometer;

a liquid reagent composition comprising luciferin, wherein the liquidreagent composition is disposed in a closed compartment, wherein the pHof the liquid reagent composition is about 6.8 or lower; and

a sampling device having a sampling portion and a handling portion;

-   -   wherein the sampling portion is adapted to acquire and        releasably retain a predetermined volume of a liquid sample in        one or more cavity that is not substantially defined by space        between a plurality of fibers;    -   wherein the sampling portion of the sampling device comprises a        dry coating that includes an effective amount of a pH-adjusting        reagent that, when contacted with the liquid reagent        composition, changes the pH of the liquid reagent composition to        6.9 or higher.

Embodiment B is an apparatus, comprising:

a container with an opening and a cuvette portion that is adapted to beoperationally coupled to a luminometer;

a liquid reagent composition comprising luciferin, wherein the liquidreagent composition is disposed in a closed compartment, wherein the pHof the liquid reagent composition is about 6.8 or lower; and

a sampling device having a sampling portion and a handling portion;

-   -   wherein the sampling portion is adapted to acquire and        releasably retain a predetermined volume of a liquid sample in        one or more cavity that is not substantially defined by space        between a plurality of fibers;    -   wherein the sampling device comprises a dry coating that        includes an effective amount of a pH-adjusting reagent that,        when contacted with the liquid reagent composition, adjusts the        pH of the liquid reagent composition to 6.9 or higher.

Embodiment C is the kit of Embodiment A or the apparatus of EmbodimentB, wherein the dry coating is disposed on the sampling portion of thesampling device.

Embodiment D is the kit or the apparatus of any one of the precedingEmbodiments, wherein the pH-adjusting reagent comprises a water-solublereagent.

Embodiment E is the kit or the apparatus of any one of the precedingEmbodiments, wherein the sampling portion comprises a calibrated loop.

Embodiment F is the kit or the apparatus of any one of the precedingEmbodiments, wherein the closed compartment comprises a frangible wall.

Embodiment G is the kit or the apparatus of Embodiment F, wherein thefrangible wall is disposed in the container between the opening and thecuvette portion.

Embodiment H is the kit of Embodiment A or the apparatus of EmbodimentB, wherein the sampling device comprises the closed compartment, whereinthe sampling device further comprises a structure that selectivelyreleases the fluid composition from the container.

Embodiment I is the kit or the apparatus of any one of the precedingEmbodiments, wherein the sampling portion comprises a foam material.

Embodiment J is the kit or the apparatus of any one of the precedingEmbodiments, wherein the pH-adjusting reagent comprises a buffercomponent selected from the group consisting of N-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid],N-[tris(hydroxymethyl)methyl]glycine, and combinations thereof.

Embodiment K is the kit or the apparatus of any one of the precedingEmbodiments, wherein the pH-adjusting reagent is coated in the one ormore cavity.

Embodiment L is the kit or the apparatus of any one of the precedingEmbodiment, wherein the coating further comprises an effective amount ofa cell extractant.

Embodiment M is the kit or the apparatus of any one of the precedingEmbodiments, wherein the luciferase enzyme consists essentially of arecombinant luciferase enzyme having luciferase activity that is lesssensitive to variations in temperature, ionic detergents, and reducingagents than a corresponding non-recombinant luciferase enzyme.

Embodiment N is the kit or the apparatus of any one of the precedingEmbodiments, wherein the coating does not comprise an effective amountof a phosphate buffer component.

Embodiment O is the kit or the apparatus of any one of the precedingEmbodiments, wherein the predetermined volume is about 0.01 millilitersto about 0.25 milliliters.

Embodiment P is a method, comprising:

using a sampling device to obtain a predetermined volume of a liquidsample;

-   -   wherein the sampling device comprises a sampling portion and a        handling portion;    -   wherein the sampling portion is adapted to acquire and        releasably retain a predetermined volume of liquid sample in one        or more cavity, wherein the one or more cavity is not        substantially defined by space between a plurality of fibers;    -   wherein the sampling device comprises a dry coating that        includes an effective amount of a pH-adjusting reagent that,        when contacted with a liquid reagent composition having a pH of        about 6.8 or lower, changes the pH of the liquid reagent        composition to 6.9 or higher;        -   wherein the liquid reagent composition comprises a            luciferin;

contacting the predetermined volume of sample and the pH-adjustingreagent with a liquid reagent composition in a container to form areaction mixture; and

using a luminometer to detect light emitted from the reaction mixture;

wherein the container comprises a cuvette portion adapted to beoperationally coupled to the luminometer.

Embodiment Q is the method of Embodiment P, further comprising the stepof agitating the predetermined volume of the sample and the liquidreagent composition in the container.

Embodiment R is the method of Embodiment P or Embodiment Q, wherein theliquid reagent composition further comprises a luciferase enzyme.

Embodiment S is the method of Embodiment R, wherein the luciferaseenzyme activity consists essentially of a recombinant luciferase enzymehaving luciferase activity that is less sensitive to variations intemperature, ionic detergents, and reducing agents than a correspondingnon-recombinant luciferase enzyme.

Embodiment T is the method of any one of Embodiments P through S,wherein contacting the sampling device comprising the sample with liquidreagent composition comprises contacting the sampling device with theliquid reagent composition at a temperature within a range of 10° C. and35° C., inclusive.

Embodiment U is the method of any one of Embodiments P though T wherein,after contacting the sampling device with liquid reagent composition, asubstantially steady-state amount of light is emitted from thecomposition in less than 20 seconds.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES

Coating Formulations:

The Coating Formulations 1 and 2 were prepared as solutions from thecomponents listed in Tables 1 and 2. The pH of Coating Formulation 1 wasabout 8.5 and the pH of Coating Formulation 2 was about 7.2.

TABLE 1 Coating Formulation 1 Weight Percentage of Component inComponent the Formulation Source Water (from a Milli Q water 96.33% EMDMillipore Corp. (Billerica, MA) purification system) Chlorhexidinedigluconate 0.32% Sigma-Aldrich Co. (St. Louis, MO) (20% in water) [CASNo. 18472-51-0] Triton X100 0.19% Sigma-Aldrich Co. (St. Louis, MO) [CASNo. 9002-93-1] Tricine 1.72% Sigma-Aldrich Co. (St. Louis, MO) [CAS No.5704-04-1] Sodium Hydroxide (4N) 1.44% VWR Corp. (Radnor, PA)

TABLE 2 Coating Formulation 2 Weight Percentage of Component inComponent the Formulation Source Water (from a Milli Q water 99.47% EMDMillipore Corp. (Billerica, MA) purification system) Chlorhexidinedigluconate 0.32% Sigma-Aldrich Co. (St. Louis, MO) (20% in water) [CASNo. 18472-51-0] Triton X100 0.21% Sigma-Aldrich Co. (St. Louis, MO) [CASNo. 9002-93-1]

Liquid Reagent Compositions:

The liquid reagent composition used in the following Examples comprisesluciferin and a luciferase enzyme. The liquid reagent composition usedin Examples 1-3 was prepared as a solution from the components listed inTable 3. The pH of the formulation was about 6.4. The total volume ofliquid reagent composition contained in the sealed cuvette portion ofeach container was about 425 microliters.

TABLE 3 Liquid reagent composition Weight Percentage of Component inComponent the Formulation Source Water (from a Milli Q water 77.6099% EMD Millipore Corp. (Billerica, MA) purification system) ADA Free Acid0.0283% Sigma-Aldrich Co. (St. Louis, MO) [CAS No. 26239-55-4] ADADisodium Salt 0.3081% Sigma-Aldrich Co. (St. Louis, MO) [CAS No.41689-31-0] EDTA Disodium Salt 0.0646% Sigma-Aldrich Co. (St. Louis, MO)Dihydrate [CAS No. 6381-92-6] Magnesium Acetate 0.1862% Sigma-AldrichCo. (St. Louis, MO) Tetrahydrate [CAS No. 16674-78-5] Sodium Azide0.0457% Sigma-Aldrich Co. (St. Louis, MO) [CAS No. 26628-22-8]D-Sorbitol 21.7106%  Molekula LTD (Gillingham, UK) [CAS No. 50-70-4]Bovine Serum Albumin 0.0457% Sigma-Aldrich Co. (St. Louis, MO) [CAS No.9048-46-8] UltraGlo Luciferase 0.0009% Promega Corp. (Madison, WI) (TypeE140X) (9 mg/L) Luciferin 0.00003-0.00004% Promega Corp. (Madison, WI)(Type XE160X) (0.3-0.4 mg/L)

The liquid reagent composition used in Comparative Examples 1-3 was thesame as listed in Table 2 with the exception that sodium hydroxide (4N)was added as a pH-adjusting reagent to adjust the pH of the liquidreagent composition from 6.4 to 6.8.

Preparation of ATP Test Apparatuses:

An apparatus similar to the design described in FIG. 1A was used. Thehandling portion of the sampling device was fabricated from apolypropylene/TPE blend. The stem and sampling portion of the samplingdevice were fabricated (i.e., molded as a unitary part) using an acetalresin. The sampling device had an overall length of about 167 mm withthe handling portion extending about 50.5 mm in length; the stemextending about 89.5 mm in length; and the sample collection ringelement extending about 27 mm in length. The cavities in the samplingportion were positioned at least about 10 mm away from the stem. Thecavities consisted of 11 spaced-apart indentations (approximately0.3-0.4 mm deep) circling the sampling portion. The cavities were spacedapart over a distance of about 14.6 mm in the sampling portion of thesampling device with a spacing of about 0.85 mm between each cavity. Asharp conical shaped tip (about 2.4 mm in length) was located at the endof the sampling portion opposite the stem, as shown in FIG. 1A.

3M Clean-Trace™ Water ATP Test devices were obtained from the 3MCompany. (St. Paul, Minn.). The pellet of freeze-dried enzyme located inthe foil-sealed compartment of each test device was removed from thecontainer and the liquid in the cuvette portion of the container wasreplaced with 425 microliters of the liquid reagent compositiondescribed in Table 3. In addition, the sampling portion of the samplingdevice was coated with one of the coating formulations described inTables 1 and 2. The sampling device was coated by holding the handlingportion of the sampling device and dipping the opposite end of thedevice into the coating solution to a point where all of the cavitieswere immersed in the coating solution. After the device was withdrawnfrom the coating solution, approximately 145 microliters of the coatingsolution remained on the sampling device. Warm air was used to dry thecoating onto the sampling device.

Example 1

The ATP test apparatus described above was used. The sampling portion ofthe sampling device was coated with Coating Formulation 1 and dried asdescribed above. The sampling device was removed from the ATP testapparatus and dipped into a vial containing 10 mL of 5×10⁻⁹ M adenosinetriphophate (ATP), commercially available from the Sigma-AldrichCorporation. While the sampling portion was immersed in the ATPsolution, gentle tapping of the sampling device against the side of thevial was used to dislodge any air bubbles from the cavities andfacilitate acquisition of the predefined volume of sample liquid. Afterabout 1-3 seconds of immersion, the sampling device was quickly removedfrom the vial and reinserted into the container. The sampling deviceretained about 145 microliters of the ATP solution. The handle of thesampling device was pressed in order to move the sampling device fromthe first operational position to the second operational position. Themovement of the sampling device in the container caused the frangibleseal to be broken and the sampling portion to be positioned in directcontact with the liquid reagent composition. The container was held inthe vertical position and shaken rapidly from side to side (pendulummotion) for 10 seconds. After shaking the container, the cuvette portionof the apparatus was immediately inserted into a Clean-Trace™ NGLuminometer (commercially available from 3M Company, St. Paul, Minn.)and measurements were recorded in relative light units (RLU). The RLUmeasurements were taken approximately every 10 seconds over a 70 secondtime period. This was accomplished by initiating a new reading for theluminometer immediately after the previous reading. Because theinstrument takes approximately 10 seconds to obtain and present each newreading, this resulted in RLU measurements taken about every 10 seconds.All testing was conducted in an environmental chamber at 10° C. Allmaterials and equipment were equilibrated in the chamber prior totesting. A total of five replicate samples were tested and the resultsare presented in Table 4.

Movement of the sampling device from the first operational position tothe second operational position resulted in the pH of the liquid reagentcomposition in the cuvette increasing from a pH approximately 6.4 to apH approximately 7.2.

Comparative Example 1

The same procedure was used as in Example 1 with the exception that thesampling device was coated with Coating Formulation 2 and the pH of theliquid reagent composition was 6.8. The RLU measurements were takenevery 10 seconds over a 70 second time period. A total of five replicatesamples were tested and the results are presented in Table 5.

TABLE 4 Sampling Device Coated with Coating Formulation 1 (Example 1)Time RLU Measurement at 10° C. (seconds) Sample 1 Sample 2 Sample 3Sample 4 Sample 5 10 13,646 16,141 13,922 12,895 17,277 20 13,682 17,54714,971 13,331 18,053 30 13,436 17,565 14,894 13,241 17,997 40 13,30317,439 14,780 13,118 17,783 50 13,220 17,219 14,623 13,033 17,578 6013,100 17,046 14,548 12,915 17,364 70 12,992 16,936 14,380 12,801 17,180

TABLE 5 Sampling Device Coated with Coating Formulation 2 (ComparativeExample 1) Time RLU Measurement at 10° C. (seconds) Sample 1 Sample 2Sample 3 Sample 4 Sample 5 10 6,667 6,048 5,049 4,200 5,046 20 8,8927,903 7,615 6,404 7,671 30 9,794 8,718 8,653 7,405 8,656 40 10,199 9,0159,114 7,904 9,107 50 10,342 9,198 9,273 8,136 9,284 60 10,395 9,1469,326 8,180 9,363 70 10,377 9,099 9,324 8,237 9,365

Example 2

The same procedure as described in Example 1 was followed with theexception that the tests were conducted at 14° C. A total of fivereplicate samples were tested and the results are presented in Table 6.

Comparative Example 2

The same procedure was used as in Example 2 with the exception that thesampling device was coated with Coating Formulation 2 and the pH of theliquid reagent composition was 6.8. The RLU measurements were takenevery 10 seconds over a 70 second time period. A total of five replicatesamples were tested and the results are presented in Table 7.

TABLE 6 Sampling Device Coated with Coating Formulation 1 (Example 2)Time RLU Measurement at 14° C. (seconds) Sample 1 Sample 2 Sample 3Sample 4 Sample 5 10 18,874 19,415 20,888 22,388 20,630 20 18,920 20,26621,430 22,749 20,624 30 18,761 20,134 21,268 22,548 20,447 40 18,53019,908 21,106 22,297 20,161 50 18,316 19,961 20,837 22,106 19,899 6018,180 19,527 20,639 21,924 19,738 70 18,040 19,352 20,666 21,776 19,597

TABLE 7 Sampling Device Coated with Coating Formulation 2 (ComparativeExample 2) Time RLU Measurement at 14° C. (seconds) Sample 1 Sample 2Sample 3 Sample 4 Sample 5 10 8,961 9,630 8,110 7,171 7,174 20 11,00311,520 10,085 9,025 9,036 30 11,628 12,065 10,537 9,574 9,504 40 11,77112,278 10,560 9,758 9,755 50 11,815 12,268 10,528 9,824 9,782 60 11,77312,260 10,486 9,821 9,824 70 11,703 12,204 10,453 9,774 9,788

Example 3

The same procedure as described in Example 1 was followed with theexception that the tests were conducted at 20° C. A total of fivereplicate samples were tested and the results are presented in Table 8.

Comparative Example 3

The same procedure was used as in Example 3 with the exception that thesampling device was coated with Coating Formulation 2 and the pH of theliquid reagent composition was 6.8. The RLU measurements were takenevery 10 seconds over a 70 second time period. A total of five replicatesamples were tested and the results are presented in Table 9.

TABLE 8 Sampling Device Coated with Coating Formulation 1 (Example 3)Time RLU Measurement at 20° C. (seconds) Sample 1 Sample 2 Sample 3Sample 4 Sample 5 10 18,605 21,358 21,553 19,799 18,697 20 18,571 21,16621,451 19,657 18,501 30 18,331 20,938 21,248 19,497 18,334 40 18,19220,788 21,141 19,367 18,179 50 18,039 20,619 20,931 19,221 18,019 6017,953 20,465 20,745 19,097 17,873 70 17,785 20,310 20,622 18,963 17,753

TABLE 9 Sampling Device Coated with Coating Formulation 2 (ComparativeExample 3) Time RLU Measurement at 20° C. (seconds) Sample 1 Sample 2Sample 3 Sample 4 Sample 5 10 11,749 11,306 10,638 11,625 10,098 2012,392 12,872 11,700 13,547 11,452 30 12,530 13,083 11,922 13,828 11,66240 12,496 13,113 11,908 13,779 11,687 50 12,382 13,039 11,903 13,77011,639 60 12,345 13,010 11,844 13,726 11,602 70 12,305 12,925 11,82713,659 11,561

Example 4

For the test samples of Examples 1-3 and the corresponding ComparativeExamples 1-3, the percent increase in the RLU measurement between thefirst time point (10 seconds) and the second time point (20 seconds) wascalculated using the following equation:

% Increase in RLU={[(RLU at 20 sec)−(RLU at 10 sec)]/(RLU at 10sec)}×100

The mean percent increase (n=5) in the RLU measurement is reported inTable 10.

TABLE 10 Mean Percent Increase in the RLU Example Number Measurement (n= 5) Standard Deviation Example 1 4.9 3.3 Comparative Example 1 43.910.8 Example 2 1.8 1.8 Comparative Example 2 23.7 2.6 Example 3 −0.7 0.3Comparative Example 3 11.8 4.2

Example 5

For the test samples of Examples 1-3 and the corresponding ComparativeExamples 1-3, the mean time (n=5) to reach the maximum RLU measurementwas determined. The results are reported in Table 11.

TABLE 11 Mean Time to the Maximum RLU Measurement in Example Numberseconds (n = 5) Example 1 22 Comparative Example 1 62 Example 2 18Comparative Example 2 48 Example 3 10 Comparative Example 3 34

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Various modifications may be made without departing from the spirit andscope of the invention. These and other embodiments are within the scopeof the following claims.

What is claimed is:
 1. A kit, comprising: a container with an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer; a liquid reagent composition comprising luciferin, wherein the liquid reagent composition is disposed in a closed compartment, wherein the pH of the liquid reagent composition is about 6.8 or lower; and a sampling device having a sampling portion; wherein the sampling portion is adapted to acquire and releasably retain a predetermined volume of a liquid sample in one or more cavity that is not substantially defined by space between a plurality of fibers; wherein the sampling device comprises a dry coating that includes an effective amount of a pH-adjusting reagent that, when contacted with the liquid reagent composition, changes the pH of the liquid reagent composition to 6.9 or higher. 